**3. Microorganisms**

Hydrogen generation in microbiological processes can be realized both by eucariota (green algae), procariota (cyanobacteria) in direct or indirect splitting of water under illumination, as well as in the fermentation and photofermentation reactions in presence of organic substances and numerous strains of bacteria. Due to very low yields of hydrogen obtained in presence of algae and cyanobacteria this paper will concentrate only on fermentative processes.

Dark fermentation process towards hydrogen is performed in presence of organothrophic bacteria. Large variety of microorganisms is involved in these reactions, therefore this paper will focus only on the description of three groups of microorganisms.

The first group belongs to anaerobic, gram-negative, mesophilic bacteria of *Clostridium* and *Bacillus* type. *C. acetobutylicum* (Chin, 2003)*, C. butyricum* (Masset, 2010, Cai, 2010) *C. pasterianum, C. bifermentants* (Wang, 2003), *C. beijerinckii* (Skonieczny, 2009)*, C.tyrobutyricum* (Jo, 2008)*, C. saccharoperbutylacetonicum* ( Alalayaha, 2008), *B. lichemiformis* (Kalia, 1994)*, B.coagulans*  (Kotay, 2007) are the most popular representatives of this group. This strains of bacteria can form spores capable to survive in extreme conditions such as low and high temperature, different pH, irradiation, extreme dry conditions or presence of deadly chemical compounds (eg. NaCl). Bacteria cells under these conditions goes to anabiosis: complete reduction of metabolic processes. The separation of already divided DNA occurs at this stage with simultaneous surrounding by two cytoplasmic membranes. The endospore formed under unfavourable conditions can return to normal activity under appropriate conditions. In this process the external protection of outer coating is destroyed. Appropriate temperature, pH and presence of feed compounds facilitate formation of vegetative cells and their growth (Setlow, 2007). In some cases presence and increase of concentration of specific compounds is the biochemical signal to stop the endospore phase. Presence of alanine, serine, cysteine together with lactic acid accelerate germination *C. botulinum* bacteria (Plowman, 2002).

molecules located at RC or LHC. The transfer of electrons from the special pair to bacteriopheophytin, located in the middle of the dielectric cytoplasmic membrane occurs in 3-4 ps. This reaction is probably intermediated by a transient product of monomeric bacteriochlorophyll BA. In the next 200 ps the electron is transferred to ubiquinone QA (connected with RC) and subsequently to ubiquinone QB. The transfer of electron to ubiquinone QB is accompanied by its protonation. The full reduction of ubiquinone QB requires two subsequent cycles in RC after which electrons finally leave RC with electrostatically neutral doubly reduced ubiquinol QH2 (Jones, 1997). The two protons required for protonation originate from cytoplasmic space. In the next step ubiquinol is oxidized by the *bc1* cytochrome complex. This complex caused reduction of the [Fe2S2] unit which is a part of cytochrome (part of Rieske unit) and releases two protons to periplasmic space. Then the cycle of electron transfer is closed by recombination of cytochrome *c2* by reduction of the special pair of bacteriochlorophylls. The cyclic transfer of electrons is accompanied by transfer of protons from cytoplasm to periplasm leading to the proton gradient between the two sides of cytoplasmic membrane, which is the most important effect of photosynthesis because it stimulates ATP synthesis and reduction of NAD+ (Vermeglio, 1999). Protons accumulated on the periplasmic space of the membrane return to the cytoplasmic space through the ATP synthase channel, which closes the transfer of

Hydrogen generation in microbiological processes can be realized both by eucariota (green algae), procariota (cyanobacteria) in direct or indirect splitting of water under illumination, as well as in the fermentation and photofermentation reactions in presence of organic substances and numerous strains of bacteria. Due to very low yields of hydrogen obtained in presence of

Dark fermentation process towards hydrogen is performed in presence of organothrophic bacteria. Large variety of microorganisms is involved in these reactions, therefore this paper

The first group belongs to anaerobic, gram-negative, mesophilic bacteria of *Clostridium* and *Bacillus* type. *C. acetobutylicum* (Chin, 2003)*, C. butyricum* (Masset, 2010, Cai, 2010) *C. pasterianum, C. bifermentants* (Wang, 2003), *C. beijerinckii* (Skonieczny, 2009)*, C.tyrobutyricum* (Jo, 2008)*, C. saccharoperbutylacetonicum* ( Alalayaha, 2008), *B. lichemiformis* (Kalia, 1994)*, B.coagulans*  (Kotay, 2007) are the most popular representatives of this group. This strains of bacteria can form spores capable to survive in extreme conditions such as low and high temperature, different pH, irradiation, extreme dry conditions or presence of deadly chemical compounds (eg. NaCl). Bacteria cells under these conditions goes to anabiosis: complete reduction of metabolic processes. The separation of already divided DNA occurs at this stage with simultaneous surrounding by two cytoplasmic membranes. The endospore formed under unfavourable conditions can return to normal activity under appropriate conditions. In this process the external protection of outer coating is destroyed. Appropriate temperature, pH and presence of feed compounds facilitate formation of vegetative cells and their growth (Setlow, 2007). In some cases presence and increase of concentration of specific compounds is the biochemical signal to stop the endospore phase. Presence of alanine, serine, cysteine

together with lactic acid accelerate germination *C. botulinum* bacteria (Plowman, 2002).

algae and cyanobacteria this paper will concentrate only on fermentative processes.

will focus only on the description of three groups of microorganisms.

protons (Paschenkoa, 2003).

**3. Microorganisms** 

The second group of fermentative bacteria active in hydrogen generation belongs to anaerobic gram-negative bacteria. The best activity in biohydrogen generation *via* dark fermentation was found for the following strains: *Enterobacter asburiae* (Jong-Hwan, 2007)*, Enterobacter cloacae* (Mandal, 2006)*, Enterobacter aerogenes* (Jo, 2008)*, Escherichia coli* (Turcot, 2008)*, Klebsiella oxytoca* (Wu, 2010) or *Citrobacter Y19* (Oha, 2003). These strains of bacteria can tolerate oxygen in environment. Here, in aerobic condition the oxygen respiration can occurs. The change of metabolic pathway provides method for survival under variable conditions of environment. These bacteria show better biological activity in comparison with those active only in completely anaerobic conditions. However, in aerobic conditions no hydrogen formation is observed. This effect is caused by inhibition of hydrogenase, enzyme catalyzing hydrogen generation.

Thermophilic bacteria operating at 60-85 oC belongs to the third group of bacteria generating hydrogen in fermentative processes (Zhang, 2003). The following strains of thermophilic bacteria of *Thermoanaerobacterium thermosaccharolyticum* (Thonga, 2008) and hyperthermophilic of *Thermatoga neapolitana* (Mars, 2010, Eriksen, 2008), *Thermococcus kodakaraensis* (Kanai, 2005)*,* or *Clostridium thermocellum* (Lewin, 2006) can generate hydrogen in presence of organic substrates at relatively high temperatures. It was established that thermophilic bacteria are the most effective from all those already described.

Application of *C. saccharolyticus* and *Thermatoga elfii* thermophilic bacteria results in 80 % yield of the theoretical one (theoretically 4 moles of glucose can be transformed into acetic acid with 100% yield) while applying saccharose or glucose (Vardar-Schara,2008), respectively. High yield in hydrogen generation is explained by Guo *et al.* (Guo, 2010) who assumes that high temperature can accelerate hydrolysis of substrates engaged in this process. At the same time Valdes-Vazquez et al. (Valdes-Vazquez, 2005) demonstrates that such results are not surprising, because optimal activity of hydrogenase is 50-70 °C. Unfortunately, the high yield of hydrogen generation with thermophilic bacteria is not equivalent to total amount of generated gas (Hallenbeck, 2009). In this situation the construction of bigger reactors is required what in consequence increase total costs. Moreover, reaction performed at higher temperatures require additional thermal energy supplied to the bioreactor.

Photofermentation in hydrogen generation is the process which requires appropriate strain of bacteria, organic substances (mainly VFA) and light with appropriate intensity. The following strains of bacteria indicate activity in photoproduction of hydrogen: *Rhodobacter sphaeroides* (Koku, 2002)*, Rhodobacter capsulatus* (Obeid, 2009), *Rhodovulum sulfidophilum*  (Maeda, 2003)*, or Rhodopseudomonas palustris* (Chen, 2008). The research of new strains active in photogeneration of hydrogen is performed in numerous laboratories all over the world. These efforts were recently awarded by discovery of activity in *Rheudopseudomanas faecalis* (Ren, 2009).

*Rhodobacter sphaeroides* belong to the group of bacteria the best recognized in hydrogen generation. These gram-negative bacteria belongs to the purple non-sulfur (PNS) *Proteobacteria* subgroup (Porter, 2008). The morphology is different because the shape of these bacteria as well as their dimensions strongly depends on the medium (see Fig. 3). In medium containing sugars the dimensions are limited to 2.0-2.5 x 2.5-3.0 μm, whereas under other conditions they can vary from 0.7 to 4.0 μm (Garrity, 2005).

Microbiological Methods of Hydrogen Generation 229

of them contain H-cluster (see Fig. 4) (Nicolet, 2000). Applying FTIR, EPR and XRD spectroscopy for analysis of monomeric hydrogenase, isolated from *Clostridium pasterianum* , it was found that H-cluster is composed from two basic units: [4Fe-4S] single group, responsible for electron transport, and the unique arrangement of [2Fe] capable to perform the reverse oxidation reaction of hydrogen. The regular cluster [4Fe-4S] is linked with four cysteine and sulfur atom of one of these forms the bridge bond between [4Fe-4S] and [2Fe]. In this dimeric system, the octahedral iron atoms are linked through two sulfur atoms (see Fig. 5) (Darensbourg, 2000). Moreover, it was found that these atoms are coordinated with five non-protein ligands (CO and CN-1) and water molecule. The bridge sulfur atoms forms additionally the 1,3- propanodithiol structure. The presence of covalent bond between sulfur

atoms influence the charge of H-cluster and electric properties (Nicolet, 2000).

Fig. 4. Scheme of iron hydrogenase in *Desulfovibrio desulfuricans (Dd)* and *Clostridium pasterianum (Cp).* F – double cluster of [4Fe-4S], L-large subunit of H-cluster, S – small subunit of H cluster, Fd- [2Fe-2S] cluster related to ferredoxin. Pink color represents the unique structure of [4Fe-4S]. In *Dd* hydrogenase large and small subunits are connected via

cysteine, whereas in Cp hydrogenase these units are linked with protein chain.

Fig. 5. Scheme of active center of iron hydrogenase (Darensbourg, 2000)

In active center of hydrogenase it is possible to identify such aminoacids as methionine and histidyne (Das, 2006). These two amino acids become attached to active center during formation of channels (for H2 and H+) connecting enzyme surface with reaction slit. The comparison of H-clusters in two strains of bacteria *Clostridium pasteurianum (Cp)* and *Desulfovibrio desulfuricans (Dd)* shows that in both cases the [2Fe] group is involved in hydrogen bond formation with lysine. However, when the second iron atom in *Cp* is

Fig. 3. *Rhodobacter sphaeroides* ATCC 17039 (Garrity, 2005).

*Rhodobacter spheroides* indicate strong chemotaxis with certain sugars, aminoacids and several organic acids (Packer, 2000). They are also capable to accept molecular nitrogen. Their metabolism is very elastic because they can germinate both in aerobic conditions (with or without light) as well as in anaerobic environment, in presence of light.

Under aerobic conditions this strain is used in purification of animal wastes (Huang, 2001) and biotransformation of toxins present in plant extracts (Yang, 2008). In the absence of oxygen *Rhodobacter spheroids* can be used in synthesis of carotenoides (Chen, 2006) and the most of all in hydrogen generation (Kars, 2010).
